2-(acetylamino)-N-benzyl-3-methoxypropanamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2016 - 28 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-benzyl-2-acetamido-3-methoxypropanamide

EC Number:

700-539-8

Cas Number:

175481-26-2

Molecular formula:

C13H18N2O3

IUPAC Name:

N-benzyl-2-acetamido-3-methoxypropanamide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Lot no.: 16-EKIN-047
- Before application of the test substance, the skin was moistened with 5 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact to the tissue

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: room temperature
- Temperature of post-treatment incubation: 37.4 - 38.5°C

REMOVAL OF TEST MATERIAL
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

MTT INTERFERENCE
The test substance was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

12.2 to 15.8 mg

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

43 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

105

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range
- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 11%
- Acceptance criteria met for variability between replicate measurements: yes, the standard deviation value of the percentage viability of three tissues treated identically was less than 17%

MTT INTERFERENCE
Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test substance did not interfere with the MTT endpoint.

Applicant's summary and conclusion

Interpretation of results:

other: GHS and CLP criteria not met

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is not irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model) performed according to OECD 439 and GLP principles, the influence of the substance on the viability of human skin was tested. Reliable positive and negative controls were included. The test substance was applied directly to 0.38 cm^2 cultured skin (12.2 to 15.8 mg). After 15 minutes, the substance was removed and cells were cultured for 43 hours. The viability of the cells was tested by reduction of MTT. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 11% whereas the test substance showed cell viability of 105%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test and the substance does not have to be classified for skin irritation according to Regulation (EC) No. 1272/2008 and GHS.